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PubMed2005

Flexible, actin-based ridges colocalise with the beta1 integrin on the surface of melanoma cells.

Using a combination of laser-scanning confocal microscopy and atomic force microscopy, we have identified flexible, actin-based structures on the surface of cells derived from the vertical growth phase of melanoma progression. These flexible structures, lacking on the surface of mature melanocytes, were observed on the surface of all four melanoma cell lines tested. Further investigation revealed that the beta1 integrin colocalises with these actin-based ridges on the cell surface, whereas beta1 integrin distribution in melanocytes did not correlate with actin-based structures. Fibronectin staining on the surface of melanoma cells was partially codistributed with the ridges. The combination of structural information derived from atomic force microscopy images and fluorescent imaging of the distribution of labelled proteins involved in invasion and metastasis has allowed us to identify a common feature that may be involved in disease progression, at the surface of vertical growth phase melanoma cells, despite the known variation in genetic composition of melanoma.

Actins↗
PubMed2001

Corneal and scleral collagens--a microscopist's perspective.

This paper reviews our existing understanding of the distribution and organisation of collagen types within the corneal and scleral stroma from a microscopical perspective. The contribution of various types of light microscopy, electron microscopy and atomic force microscopy to this field are separately discussed. Light microscopy was used in the earliest studies of the cornea and lead to the first description of the lamellar structure of the stroma. More recently polarised light microscopy has been used to obtain specific information on fibril orientation within individual lamellae. Light microscope immunolabelling techniques have been utilised to determine the distribution of several collagen types within the cornea and sclera, while recent developments in confocal microscopy have allowed detailed observations to be made on live cornea. Scanning electron microscopy has proved useful in determining the 3D organisation of lamellae within both corneal and scleral stroma. The transmission electron microscope was responsible for first revealing the regular diameter and high degree of order of the collagen fibrils present in the corneal stroma and contrasting this with the irregular diameter of fibrils present in sclera. This finding lead directly to the formulation of a theory of corneal transparency based on the uniformity of fibril diameter and packing. The use of specialised stains such as cuprolinic blue allowed direct observation of the glycosaminoglycan chains on proteoglycan molecules in cornea and sclera. These images allowed the binding sites of the proteoglycans along the collagen fibrils to be identified and provided convincing evidence for the importance of the proteoglycan molecules in collagen fibril organisation. Immunogold labelling has been used to map the distribution of several collagen types within the corneal and scleral stroma at the ultrastructural level and provided critical evidence for the role of type V collagen in the regulation of fibril diameter within the cornea. Specialised freezing-etching techniques have revealed the surface features of the collagen fibrils in corneal stroma, indicating clearly the presence of crossbridge structures between fibrils. The technique of rotary shadowing has been used to determine the conformation of several collagen types. In more recent years atomic force microscopy has been applied to the study of the corneal stroma. It has largely confirmed the observations made by the transmission electron microscope and provided independent evidence of crossbridge structures between the collagen fibres in cornea and sclera. The full potential of this technique has yet to be realised.

Animals↗
PubMed2001

Two-dimensional crystals: a powerful approach to assess structure, function and dynamics of membrane proteins.

Electron crystallography and atomic force microscopy allow the study of two-dimensional membrane protein crystals. While electron crystallography provides atomic scale three-dimensional density maps, atomic force microscopy gives insight into the surface structure and dynamics at sub-nanometer resolution. Importantly, the membrane protein studied is in its native environment and its function can be assessed directly. The approach allows both the atomic structure of the membrane protein and the dynamics of its surface to be analyzed. In this way, the function-related conformational changes can be assessed, thus providing a detailed insight on the molecular mechanisms of essential biological processes.

Cell Membrane↗
PubMedDate not supplied

Characterization of cellulose whiskers and their nanocomposites by atomic force and electron microscopy.

The aim of this work was to compare and explore electron microscopy and atomic force microscopy (AFM) for structure determination of cellulose whiskers and their nanocomposite with poly(lactic acid). From conventional bright-field transmission electron microscopy (TEM) it was possible to identify individual whiskers, which enabled determination of their sizes and shape. AFM overestimated the width of the whiskers due to the tip-broadening effect. Field emission scanning electron microscopy (FESEM) allowed for a quick examination giving an overview of the sample; however, the resolution was considered insufficient for detailed information. Ultramicrotomy of nanocomposite films at cryogenic temperatures enabled detailed inspection of the cellulose whiskers in the poly(lactic acid) matrix by AFM. FESEM applied on fractured surfaces allowed insight into the morphology of the nanocomposite, although rather restricted due to the metal coating and limited resolution. Detailed information was obtained from TEM; however, this technique required staining and suffered in general from limited contrast and beam sensitivity of the material.

Biocompatible Materials↗
PubMed2002

Chair-boat transitions in single polysaccharide molecules observed with force-ramp AFM.

Under a stretching force, the sugar ring of polysaccharide molecules switches from the chair to the boat-like or inverted chair conformation. This conformational change can be observed by stretching single polysaccharide molecules with an atomic force microscope. In those early experiments, the molecules were stretched at a constant rate while the resulting force changed over wide ranges. However, because the rings undergo force-dependent transitions, an experimental arrangement where the force is the free variable introduces an undesirable level of complexity in the results. Here we demonstrate the use of force-ramp atomic force microscopy to capture the conformational changes in single polysaccharide molecules. Force-ramp atomic force microscopy readily captures the ring transitions under conditions where the entropic elasticity of the molecule is separated from its conformational transitions, enabling a quantitative analysis of the data with a simple two-state model. This analysis directly provides the physico-chemical characteristics of the ring transitions such as the width of the energy barrier, the relative energy of the conformers, and their enthalpic elasticity. Our experiments enhance the ability of single-molecule force spectroscopy to make high-resolution measurements of the conformations of single polysaccharide molecules under a stretching force, making an important addition to polysaccharide spectroscopy.

Elasticity↗
PubMed2000

Surface characterization of aspirin crystal planes by dynamic chemical force microscopy.

Tapping mode (TM) atomic force microscopy (AFM) has been applied in a novel fashion to characterize and distinguish the (001) and (100) surfaces of individual aspirin crystals. The surface characterization was achieved by amplitude-phase, distance (a-p,d) measurements employing gold-coated AFM probes functionalized with self-assembled monolayers (SAM). Experiments using model probes coated with -CH3 and -COOH terminated SAMs have been performed on the two aspirin crystal planes (001) and (100). Results indicate that the hydrophobic -CH3 terminated AFM probes had a greater degree of interaction with the crystal plane (001), whereas the -COOH terminated AFM probes had a larger interaction with the crystal plane (100). Interpretation of these data, based upon the chemistries of the probes, correlates with current understanding of the crystal surface chemistry derived from X-ray diffraction data and dissolution rate studies.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2005

Simultaneous force and fluorescence measurements of a protein that forms a bond between a living bacterium and a solid surface.

All microbial biofilms are initiated through direct physical contact between a bacterium and a solid surface, a step that is controlled by inter- and intramolecular forces. Atomic force microscopy and confocal laser scanning microscopy were used simultaneously to observe the formation of a bond between a fluorescent chimeric protein on the surface of a living Escherichia coli bacterium and a solid substrate in situ. The chimera was composed of a portion of outer membrane protein A (OmpA) fused to the cyan-fluorescent protein AmCyan. Sucrose gradient centrifugation and fluorescent confocal slices through bacteria demonstrated that the chimeric protein was targeted and anchored to the external cell surface. The wormlike chain theory predicted that this protein should exhibit a nonlinear force-extension "signature" consistent with the sequential unraveling of the AmCyan and OmpA domains. Experimentally measured force-extension curves revealed a unique pair of "sawtooth" features that were present when a bond formed between a silicon nitride surface (atomic force microscopy tip) and E. coli cells expressing the OmpA-AmCyan protein. The observed sawtooth pair closely matched the wormlike chain model prediction for the mechanical unfolding of the AmCyan and OmpA substructures in series. These sawteeth disappeared from the measured force-extension curves when cells were treated with proteinase K. Furthermore, these unique sawteeth were absent for a mutant stain of E. coli incapable of expressing the AmCyan protein on its outer surface. Together, these data show that specific proteins exhibit unique force signatures characteristic of the bond that is formed between a living bacterium and another surface.

Bacterial Adhesion↗
PubMed2005

Properties of two-component Langmuir monolayer of single chain perfluorinated carboxylic acids with dipalmitoylphosphatidylcholine (DPPC).

The surface pressure (pi)- and the surface potential (DeltaV)-area (A) isotherms were obtained for two-component monolayers of four different perfluorocarboxylic acids (FCns; perfluorododecanoic acid: FC12, perfluorotetradecanoic acid: FC14, perfluorohexadecanoic acid: FC16, perfluorooctadecanoic acid: FC18) with dipalmitoylphosphatidylcholine (DPPC) on substrate solution of 0.15 M NaCl (pH 2.0) at 298.2 K as a function of compositions in the mixtures by employing the Wilhelmy method, the ionizing electrode method, the fluorescence microscopy, and the atomic force microscopy. The data for the two-component monolayers on these systems were analyzed in terms of the additivity rule. Assuming a regular surface mixture, the Joos equation which allows one to describe the collapse pressure of a two-component monolayer with miscible components was used to declare the miscibility of the monolayer state, and an interaction parameter and an interaction energy were calculated. The new finding was that FCns and DPPC are miscible or immiscible depending on chain length increment of fluorocarbon part. That is, FC12/DPPC monolayer was perfectly miscible, and FC14/DPPC, and FC16/DPPC (0 < or = X(FC16) < or = 0.3) monolayers were partially miscible. While FC16/DPPC (0.3 < X(FC16) < 1) and FC18/DPPC systems are immiscible in the monolayer state. Furthermore, the mean molecular area, the surface dipole moment, and the phase diagrams enabled us to estimate the molecular orientation of four different perfluorocarboxylic acids/DPPC in the two-component monolayer state. One type of phase diagrams was obtained and classified into the positive azeotropic type. The miscibility of FCns and DPPC in the monolayer was also supported by fluorescence microscopy and atomic force microscopy. FC12/DPPC, FC14/DPPC and FC16/DPPC (0 < or = X(FC16) < or = 0.3) two-component monolayers on 0.15 M NaCl (pH 2) showed that FC12, FC14 and FC16 (0 < or = X(FC16) < or = 0.3) can dissolve or partially dissolve the ordered solid DPPC domains formed upon compression. This indicates that these fluorinated amphiphiles soften or harden the lipid depending on their chain length.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2000

Nanoscale topography of the corneal epithelial basement membrane and Descemet's membrane of the human.

PURPOSE: Quantitatively define and compare the nanoscale topography of the corneal epithelial basement membrane (anterior basement membrane) and Descemet's membrane (posterior basement membrane) of the human. METHODS: Human corneas not suitable for transplantation were obtained from the Wisconsin Eye Bank. The corneas were placed in 2.5 mM EDTA for 2.5 h or 30 min. for removal of the epithelium or endothelium, respectively. After removal of the overlying cells, specimens were fixed in 2% glutaraldehyde and either examined in this state by atomic force microscopy only or dehydrated through an ethanol series and prepared for transmission electron microscopy (TEM), scanning electron microscopy (SEM), and atomic force microscopy (AFM). RESULTS: The subepithelial and subendothelial basement membrane surfaces have a similar appearance that consists of an interwoven meshwork of fibers and pores. Topographic feature sizes were found to be in the nanometer size range with the epithelial basement membrane features larger and less densely packed than Descemet's membrane features. The topographic features are fractile in nature and increase surface area for cell contact. CONCLUSION: With the use of the TEM, SEM, and AFM, a detailed description of the surface topography of corneal epithelial basement membrane and Descemet's membrane of the human cornea are provided. The significance of differences in corneal basement membrane topography may reflect differences in function of the overlying cells or may be related to differences in cell migration and turnover patterns between the epithelium and endothelium.

Aged↗
PubMed1996

Covalent immobilization of native biomolecules onto Au(111) via N-hydroxysuccinimide ester functionalized self-assembled monolayers for scanning probe microscopy.

We have worked out a procedure for covalent binding of native biomacromolecules on flat gold surfaces for scanning probe microscopy in aqueous buffer solutions and for other nanotechnological applications, such as the direct measurement of interaction forces between immobilized macromolecules, of their elastomechanical properties, etc. It is based on the covalent immobilization of amino group-containing biomolecules (e.g., proteins, phospholipids) onto atomically flat gold surfaces via omega-functionalized self-assembled monolayers. We present the synthesis of the parent compound, dithio-bis(succinimidylundecanoate) (DSU), and a detailed study of the chemical and physical properties of the monolayer it forms spontaneously on Au(111). Scanning tunneling microscopy and atomic force microscopy (AFM) revealed a monolayer arrangement with the well-known depressions that are known to stem from an etch process during the self-assembly. The total density of the omega-N-hydroxysuccinimidyl groups on atomically flat gold was 585 pmol/cm(2), as determined by chemisorption of (14)C-labeled DSU. This corresponded to approximately 75% of the maximum density of the omega-unsubstituted alkanethiol. Measurements of the kinetics of monolayer formation showed a very fast initial phase, with total coverage within 30 S. A subsequent slower rearrangement of the chemisorbed molecules, as indicated by AFM, led to a decrease in the number of monolayer depressions in approximately 60 min. The rate of hydrolysis of the omega-N-hydroxysuccinimide groups at the monolayer/water interface was found to be very slow, even at moderately alkaline pH values. Furthermore, the binding of low-molecular-weight amines and of a model protein was investigated in detail.

Enzymes, Immobilized↗
PubMed2006

Characterizing folding, structure, molecular interactions and ligand gated activation of single sodium/proton antiporters.

Using the example of sodium/proton antiporter from Escherichia coli NhaA, we review the capabilities of single-molecule atomic force microscopy and force spectroscopy to observe structural and functional insights of a membrane protein, which are not attainable by other traditional methods. While atomic force microscopy provides high-resolution topographs of single membrane proteins, their oligomeric state and assembly, single-molecule force spectroscopy experiments detect molecular interactions of the protein. The sensitivity of this method makes it possible to detect and locate interactions that stabilize secondary structures such as transmembrane alpha-helices, polypeptide loops and segments within them. Controlled refolding experiments using single-molecule force spectroscopy observed individual secondary structure segments folding into the functional protein. Various folding pathways of NhaA were detected, each one exhibiting a certain probability to be taken. Time-lapse refolding experiments enabled determining the folding kinetics and hierarchy of individual secondary structural elements. Recent examples detected and located the ligand binding of an antiporter. Similarly, inhibitor binding and location can be detected which in future may guide towards comparative studies of agonist and antagonist altering the functional state of a membrane protein. We review current and future potentials of these approaches to characterize the action of pharmacological molecules on the antiporter function.

Amino Acid Sequence↗
PubMed2005

Single-molecule measurements of the persistence length of double-stranded RNA.

Over the past few years, it has become increasingly apparent that double-stranded RNA (dsRNA) plays a far greater role in the life cycle of a cell than previously expected. Numerous proteins, including helicases, polymerases, and nucleases interact specifically with the double helix of dsRNA. To understand the detailed nature of these dsRNA-protein interactions, the (bio)chemical, electrostatic, and mechanical properties of dsRNA need to be fully characterized. We present measurements of the persistence length of dsRNA using two different single-molecule techniques: magnetic tweezers and atomic force microscopy. We deduce a mean persistence length for long dsRNA molecules of 63.8 +/- 0.7 nm from force-extension measurements with the magnetic tweezers. We present atomic force microscopy images of dsRNA and demonstrate a new method for analyzing these, which yields an independent, yet consistent value of 62 +/- 2 nm for the persistence length. The introduction of these single-molecule techniques for dsRNA analysis opens the way for real-time, quantitative analysis of dsRNA-protein interactions.

Biophysics↗
PubMed2002

Fabrication of ordered arrays of InP microstructures by wet chemical etching with Au masks.

Ordered arrays of InP microstructures have been fabricated on InP(001) substrates by wet chemical etching in aqueous HCl with patterned Au masks. The masks were produced by Au deposition through copper grids or a monolayer of polystyrene microspheres. Square InP mesas (20 x 20 microns) and pillars (approximately 100 nm in both diameter and height) were both produced and characterized by scanning electron microscopy and atomic force microscopy.

Crystallization↗
PubMed2003

Pathway shifts and thermal softening in temperature-coupled forced unfolding of spectrin domains.

Pathways of unfolding a protein depend in principle on the perturbation-whether it is temperature, denaturant, or even forced extension. Widely-shared, helical-bundle spectrin repeats are known to melt at temperatures as low as 40-45 degrees C and are also known to unfold via multiple pathways as single molecules in atomic force microscopy. Given the varied roles of spectrin family proteins in cell deformability, we sought to determine the coupled effects of temperature on forced unfolding. Bimodal distributions of unfolding intervals are seen at all temperatures for the four-repeat beta(1-4) spectrin-an alpha-actinin homolog. The major unfolding length corresponds to unfolding of a single repeat, and a minor peak at twice the length corresponds to tandem repeats. Increasing temperature shows fewer tandem events but has no effect on unfolding intervals. As T approaches T(m), however, mean unfolding forces in atomic force microscopy also decrease; and circular dichroism studies demonstrate a nearly proportional decrease of helical content in solution. The results imply a thermal softening of a helical linker between repeats which otherwise propagates a helix-to-coil transition to adjacent repeats. In sum, structural changes with temperature correlate with both single-molecule unfolding forces and shifts in unfolding pathways.

Circular Dichroism↗
PubMed2003

Packaging of single DNA molecules by the yeast mitochondrial protein Abf2p.

Mitochondrial and nuclear DNA are packaged by proteins in a very different manner. Although protein-DNA complexes called "nucleoids" have been identified as the genetic units of mitochondrial inheritance in yeast and man, little is known about their physical structure. The yeast mitochondrial protein Abf2p was shown to be sufficient to compact linear dsDNA, without the benefit of supercoiling, using optical and atomic force microscopy single molecule techniques. The packaging of DNA by Abf2p was observed to be very weak as evidenced by a fast Abf2p off-rate (k(off) = 0.014 +/- 0.001 s(-1)) and the extremely small forces (<0.6 pN) stabilizing the condensed protein-DNA complex. Atomic force microscopy images of individual complexes showed the 190-nm structures are loosely packaged relative to nuclear chromatin. This organization may leave mtDNA accessible for transcription and replication, while making it more vulnerable to damage.

Binding Sites↗
PubMed2004

Molecular dynamics simulations of forced conformational transitions in 1,6-linked polysaccharides.

Recent atomic force microscopy stretching measurements of single polysaccharide molecules suggest that their elasticity is governed by force-induced conformational transitions of the pyranose ring. However, the mechanism of these transitions and the mechanics of the pyranose ring are not fully understood. Here we use steered molecular dynamics simulations of the stretching process to unravel the mechanism of forced conformational transitions in 1,6 linked polysaccharides. In contrast to most sugars, 1,6 linked polysaccharides have an extra bond in their inter-residue linkage, C5-C6, around which restricted rotations occur and this additional degree of freedom increases the mechanical complexity of these polymers. By comparing the computational results with the atomic force microscopy data we determine that forced rotations around the C5-C6 bond have a significant and different impact on the elasticity of alpha- and beta-linked polysaccharides. Beta-linkages of a polysaccharide pustulan force the rotation around the C5-C6 bonds and produce a Hookean-like elasticity but do not affect the conformation of the pyranose rings. However, alpha-linkages of dextran induce compound conformational transitions that include simultaneous rotations around the C5-C6 bonds and chair-boat transitions of the pyranose rings. These previously not-recognized transitions are responsible for the characteristic plateau in the force-extension relationship of dextran.

Carbohydrate Conformation↗
PubMed2004

Force spectroscopy of the double-tethered concanavalin-A mannose bond.

We present the measurement of the force required to rupture a single protein-sugar bond using a methodology that provides selective discrimination between specific and nonspecific binding events and helps verify the presence of a single functional molecule on the atomic force microscopy tip. In particular, the interaction force between a polymer-tethered concanavalin-A protein (ConA) and a similarly tethered mannose carbohydrate was measured as 47 +/- 9 pN at a bond loading rate of approximately 10 nN/s. Computer simulations of the polymer molecular configurations were used to determine the angles that the polymers could sweep out during binding and, in conjunction with mass spectrometry, used to separate the angular effects from the effects due to a distribution of tether lengths. We find that when using commercially available polymer tethers that vary in length from 19 to 29 nm, the angular effects are relatively small and the rupture distributions are dominated by the 10-nm width of the tether length distribution. In all, we show that tethering both a protein and its ligand allows for the determination of the single-molecule bond rupture force with high sensitivity and includes some validation for the presence of a single-tethered functional molecule on the atomic force microscopy tip.

Computer Simulation↗
PubMed2005

Quantifying the specific binding between West Nile virus envelope domain III protein and the cellular receptor alphaVbeta3 integrin.

A previous study has illustrated that the alphaVbeta3 integrin served as the functional receptor for West Nile virus (WNV) entry into cells. Domain III (DIII) of WNV envelope protein (E) was postulated to mediate virus binding to the cellular receptor. In this study, the specificity and affinity binding of WNV E DIII protein to alphaVbeta3 integrin was confirmed with co-immunoprecipitation and receptor competition assay. Binding of WNV E DIII protein to alphaVbeta3 integrin induced the phosphorylation of focal adhesion kinase that is required to mediate ligand-receptor internalization into cells. A novel platform was then developed using the atomic force microscopy to measure this specific binding force between WNV E DIII protein and the cellular receptor, alphaVbeta3 integrin. The single protein pair-interacting force measured was in the range of 45 +/- 5 piconewtons. This interacting force was highly specific as minimal force was measured in the WNV E DIII protein interaction with alphaVbeta5 integrin molecules and heparan sulfate. These experiments provided an insight to quantitate virus-receptor interaction. Force measurement using atomic force microscopy can serve to quantitatively analyze the effect of candidate drugs that modulate virus-host receptor affinity.

Cell Line, Tumor↗